A novel trophectoderm biopsy technique for all blastocyst stages

Abstract Purpose This study was conducted to assess the effectiveness of a new trophectoderm (TE) biopsy method that does not require prior opening of the zona pellucida at the blastocyst stage. Methods TE biopsy was conducted using a modified extrusion method for embryos during the cleavage stage. In this method, culture medium was injected into the perivitelline space to help extrude TE cells from the zona pellucida before TE biopsy. Results Our extrusion method preserves the embryo culture environment until immediately before biopsy because it does not require opening of the zona pellucida prior to TE biopsy. Furthermore, this method does not require a waiting time for blastocyst hatching after laser irradiation, thereby minimizing damage to the embryos and maintaining the time schedule of culture operations. Conclusions TE biopsy using this novel extrusion method may be useful in various applications, including the collection of TE cells for next‐generation sequencing analysis.


| INTRODUC TI ON
To increase the success rate of in vitro fertilization, the rate of implantation of embryos harboring numerical chromosomal abnormalities must be limited while ensuring implantation of euploid embryos.
Currently, the most effective method for selecting euploid embryos involves preimplantation genetic testing for aneuploidy (PGT-A).
Considering that 60% of spontaneous abortions and stillbirths result from aneuploidy, testing for these chromosomal abnormalities can greatly reduce the rate of pregnancy loss. 1 Specifically, the reported correlation among the incidence of miscarriage, aneuploid embryos, and maternal age at delivery highlights the effectiveness of PGT-A screening. 2 However, PGT-A requires the collection of biopsied cells from preimplantation embryos. Although conventional biopsies are commonly performed on embryos during the cleavage stage, this strategy only permits the examination of one or two cells, leading to an increased risk of misdiagnosis because of the high incidence of embryo mosaicism at the cleavage stage. 3 Another disadvantage of this method is the maldevelopment of embryos, with an associated low pregnancy rate as a result of a low ratio of the number of biopsy specimens to total cells. 4,5 Because of this, our hospital has adopted a modified extrusion method (m-Ext method) involving a modified version of the original extrusion method (o-Ext method) 6 for the biopsy of embryo blastomeres during the cleavage stage of development; this method is combined with fluorescence in-situ hybridization to diagnose aneuploid embryos. Moreover, recent advances in genetic analysis techniques have led to the combination of array-comparative genomic hybridization, next-generation sequencing (NGS), and PGT of trophectoderm (TE) cells in blastocysts, with most recent approaches shifting toward NGS. Additionally, the use of TE biopsies at the blastocyst stage allows for multiple collections of cell specimens.
The m-Ext method involves creating an opening in the zona pellucida by laser irradiation. Culture medium is then injected to the perivitelline space to extrude blastomeres, which eject from the opening point used for culture injection. Thus, the m-Ext method differs from the o-Ext method, i.e., the former has the same injection and ejection points of medium and blastomeres, respectively, while the latter has different injection and ejection points ( Figure 1). In this study, we have used a novel extrusion method (n-Ext) for embryos that had developed to blastocysts without prior zona pellucida opening, which is used for biopsy at the cleavage stage. We compared this method to the conventional TE biopsy method for embryos developing into hatching blastocysts by opening the zona pellucida in advance.

| Ovarian stimulation
Oocyte retrieval was performed at the Takeuchi Ladies Clinic between January 2016 and January 2020. During the oocyte retrieval cycle, ovarian stimulation was performed using standard gonadotropin-releasing hormone agonist follicle-stimulating hor-

| Embryo culture
Cumulus oocyte complexes obtained using the oocyte retrieval procedure described above were preincubated for 3 h (in a gas phase of 5% O 2 , 6% CO 2 , and 89% N 2 at 37°C), followed by denuding of cumulus/oocyte complex cells. Oocytes in metaphase II were used for intracytoplasmic sperm injection and then cultured in an EmbryoScope + time-lapse incubator (Vitrolife). Fertilization was confirmed 16-18 h post-intracytoplasmic sperm injection, after which blastocysts developed over a maximum 6-day incubation period in Global medium (LG; Life Global) while covered with mineral oil (SAGE, Origio). Biopsies were performed on blastocysts that patients had elected to destroy. Corning, Inc.). The biopsy pipette was washed with polyvinyl pyrrolidone (Orgio) to coat the inside of the pipette and prevent cell attachment. Two types of conventional biopsy methods were performed. The first involved creating an opening in the zona pellucida of the embryos before they had reached the blastocyst stage on day 3 or 4. The second method involved artificially shrinking the blastocyst 7 to widen the perivitelline space. Next, laser irradiation was applied partially to the zona pellucida. In both methods, embryos that reached the blastocyst stage were fixed at the 9 o'clock position using the holding pipette, and TE cells were collected, avoiding the ICM. Unlike biopsies of embryos at the cleavage stage, biopsies at the blastocyst stage, during which cells are adjacent to each other, commonly involve cutting of the TE cells by laser irradiation after suctioning the required TE cells into the biopsy pipette (pulling-stretching method) or using a scraping motion against the holding pipette (flicking method). In this study, we primarily employed the pulling-stretching method for laser irradiation.

| Laser irradiation of the zona pellucida
The creation of an opening in the zona pellucida, biopsy, and assisted hatching (AHA) were performed using the LYKOS laser perforation system (Hamilton Thorne, Inc.). Creation of an opening in the zona pellucida was performed using laser irradiation with a heat range (pulse of 300 μs) that minimized damage to the cells.

| Clinical application of the n-Ext method in TE biopsy
Biopsies performed using the n-Ext method can be conducted without creating an opening in the zona pellucida prior to extrusion and on non-hatching blastocysts. In embryos suspended with the holding pipette, a small opening was created in the zona pellucida (at a site containing more TE cells) via laser irradiation (90 μs) from the 3 o'clock position. Culture medium was injected into the perivitelline space through the biopsy pipette to remove TE cells from the inner surface of the zona pellucida. The small opening of the zona pellucida was gradually expanded by applying laser irradiation (120 μs) until the diameter was approximately equivalent to that of the biopsy pipette. Culture medium was then injected into the zona pellucida to extrude TE cells from the opening, which were collected as described for the conventional method ( Figure 2).

| Tubing and NGS analysis of cell specimens obtained in TE biopsy
Tubing and NGS analysis of the cell specimens obtained by TE biopsy were performed as described by Takeuchi et al. 8

| Statistical analysis
We used BellCurve (version 3.20) for Excel (Social Survey Research Information Co., Ltd) for all statistical analyses. Statistical analyses were performed using the Kruskal-Wallis analysis of variance (Table 1). A p-value <0.05 was considered to indicate statistically significant results.

| RE SULTS
When comparing the required operation time for collecting biopsies between the two methods, the protocol for the n-Ext method was significantly longer (129.25 ± 37.30 s) compared to the conventional method (64.00 ± 17.58 s) (p < 0.05). There was no significant difference in maternal age (41.17 ± 3.54 vs. 42.00 ± 2.68 years, respectively) or the number of collected cells (5.69 ± 1.60 vs. 6.00 ± 1.41, respectively) between the two groups. After biopsy, all blastocysts were viable (Table 1). Furthermore, when performing NGS analysis on samples obtained via the n-Ext method, all cells were found to be stable (Table 2). Representative charts are presented in Figure 3.
The mean number of cells collected by biopsy using the conventional method was 5.69 ± 1.60, which is similar to the 4-5 cells collected by Kokkali et al. 9 using this method, whereas that in the biopsy using the n-Ext method was 6.00 ± 1.41 in the comparative (conventional vs. novel) study and 5.87 ± 1.88 for NGS analysis.

| DISCUSS ION
Unlike the conventional method, which can only be applied for the biopsy of hatching blastocysts, the n-Ext method is advantageous because it can be used for the biopsy of expanded blastocysts before hatching.
There are two types of conventional methods for blastocyst biopsy (i.e., methods for creating an opening in the zona pellucida before TE biopsy); the first involves creating an opening in the zona pellucida of embryos before the blastocyst stage (day 3 or 4), at which point it is difficult to identify the precise position of the ICM, and therefore, this method is associated with an increased risk of ICM hatching, making it challenging to perform this method accurately. The second method involves artificial shrinkage and irradiation of the zona pellucida, thereby necessitating recovery culture. In contrast, TE biopsy using the n-Ext method does not require prior creation of an opening in the zona pellucida, eliminating the need for recovery culture. This means that TE biopsy can be performed without waiting for the blastocysts to become available for biopsy (blastocyst hatching). Typical recovery for blastocyst hatching requires 2-3 h, providing a schedule that is more suitable for subsequent procedures (Figure 4). Moreover, TE biopsy using the n-Ext method may reduce unnecessary observations, thereby maintaining the culture environment and minimally affecting the success of subsequent steps, besides biopsy.
The results of NGS analysis also showed that the n-Ext method ensures the collection of sufficient cells for further analysis.
However, this method also required an extended duration to complete the biopsy procedure (approximately 2 min) compared with the conventional method, as collection of TE biopsies before blastocyst hatching takes a longer time period. In contrast, biopsy collection using the conventional extrusion method is relatively short (approximately 1 min). However, the conventional method also requires 1 min to create an opening in the zona pellucida prior to the hatching process. Hence, the overall in vitro exposure times between the two methods were comparable. Another disadvantage of the novel extrusion method is that blastocysts occasionally shrink during the operation. In such cases, the biopsy must be completed before complete shrinkage by inserting the pipette deep into the zona pellucida.
Rubino et al. 10   freeze-thaw cycle after biopsy. However, if the procedure required for collecting a biopsy from the embryos is highly invasive, blastocysts may not recover after thawing. To assess the viability of blastocysts after being subjected to the n-Ext biopsy method, we previously performed a biopsy on frozen-thawed blastocysts using this method. The blastocysts were refrozen and thawed using a CryoTip, leading to the recovery of all blastocysts. 8 In the present study, all blastocysts survived after biopsy (Table 1). Additionally, there was no decrease in the grade of the blastocysts after recovery culture. These results demonstrate that biopsies performed via the novel extrusion method did not affect the recovery of frozenthawed blastocysts. Furthermore, a good pregnancy rate has been reported with refrozen-thawed embryos using a CryoTip, 11 suggesting the usefulness of performing biopsy using the n-Ext method and freeze-thawing with a CryoTip.
In this study, the number of cells varied from three to nine. One of the possible reasons for three-cell collection was that the TE grade was C. Because embryo survival is lower after biopsy when taking Several studies also demonstrated the effectiveness of using TE biopsy to improve pregnancy rates. [17][18][19] Preventing aneuploid embryo implantation effectively reduces the risk of unnecessary abortions, leading to fewer treatment cycles and reduced costs, highlighting the advantages of PGT. [20][21][22] Taken together, our results demonstrate that biopsies performed using the novel extrusion method minimize the impact on daily operations by eliminating the need for recovery culture. In addition, this method enables collection of biopsies during the blastocyst stage before hatching, as it does not required prior laser irradiation.
Therefore, biopsies using the novel extrusion method may facilitate the collection of optimal cells required for NGS analysis to ensure accurate and stable results.

ACK N OWLED G EM ENTS
The authors thank the embryologist doctors and nursing staff at Takeuchi Ladies Clinic/Center for Reproductive medicine (Aira-shi, Kagoshima, Japan) for providing technical assistance and support.
We also thank Editage (www.edita ge.com) for English language editing.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
The study was performed with the approval of the institutional re-